Molecular identification of Phlebotomus kandelakii apyrase and assessment of the immunogenicity of its recombinant protein in BALB/c mice

Sand fly salivary proteins have immunomodulatory and anti-inflammatory features; hence, they are proven to perform important roles in the early establishment of Leishmania parasite in the vertebrate host. Among them, salivary apyrase with anti-hemostatic properties has a crucial role during the blood meal process. In the present study, a Genome-Walking method was used to characterize a full-length nucleotide sequence of Phlebotomus (P.) kandelakii apyrase (Pkapy). Bioinformatics analyses revealed that Pkapy is a ~ 36 kDa stable and hydrophilic protein that belongs to the Cimex family of apyrases. Moreover, recombinant proteins of Pkapy and P. papatasi apyrase (Ppapy) were over-expressed in Escherichia coli BL2 (DE3) and their antigenicity in BALB/c mice was evaluated. Dot-blot and ELISA results indicated that both recombinant apyrases could induce antibodies in BALB/c. Moreover, a partial cross-reactivity between Pkapy and Ppapy was found. In vitro stimulation of splenocytes from immunized mice with the recombinant proteins indicated cross-reactive T cell proliferative responses. Cytokine analysis revealed significant production of IFN-γ (p < 0.001) and IL-10 (p < 0.01) in response to Pkapy. In conclusion, the full-length nucleotide sequence and molecular characteristics of Pkapy were identified for the first time. Immunologic analyses indicated that Pkapy and Ppapy are immunogenic in BALB/c mice and show partial cross-reactive responses. The immunity to Pkapy was found to be a Th1-dominant response that highlights its potential as a component for an anti-Leishmania vaccine.

Pkapy was estimated to be approximately 400 bp at the 3′-end and 150 bp at the 5′-end. Therefore, the amplicons of more than 400 bp (i.e., 3 amplicons in GWA, 1 amplicon in GWF, and 1 amplicon in GWG) were selected for the downstream sequence assessment (Additional file 1: Fig. S1a), and amplicons of more than 150 bp (i.e., 2 amplicons in GWA for upstream sequence determination; Additional file 1: Fig. S1b) were selected and extracted from the agarose gel and were subjected for TA cloning and nucleotide sequencing. The retrieved sequences were revealed to belong to the apyrase gene family by nucleotide BLAST. These sequences were assembled onto the middle part sequence using the overlapping regions. Consequently, the full assembled sequence of Pkapy gene was submitted to GenBank (Accession N o .: MN893300).
In silico findings and predictions. As indicated in Table 2 and according to the performed computations, physicochemical parameters, namely Mw and pI of Pkapy in the mature form were predicted to be 35.2 kDa and 9.2, respectively. The protein was composed of 309 amino acids, containing 35 negatively-charged (Asp + Glu), and 43 positively-charged residues (Arg + Lys). Based on the aliphatic and instability indices (79.55 and 18.48, respectively), Pkapy was predicted to be a stable protein. Moreover, the grand average of hydropathicity (GRAVY) of the predicted protein was − 0.49 which indicates its hydrophilic nature. Prediction of N-and O-glycosylation Table 1. List of Genome-walking primers (a) and PCR programs (b).    Table 2). The secondary structure analysis of Pkapy indicated that the protein was made up of alpha-helix (7.77%), extended strand (39.81%), and random coil (52.43%). Since 162 of 309 amino acid residues were found to be localized to the random coil, this would probably be the main secondary structure of Pkapy (Table 2). Physicochemical properties and secondary structure prediction of Ppapy are also presented in Table 2. Signal peptide cleavage sites were predicted for Ppapy and Pkapy amino acid sequences between residues 21-22 and 20-21 with 0.9614 and 0.9468 probabilities, respectively.
Protein sequence alignment comparison between Pkapy (QNG40038.1) with apyrases from a few sand fly species and other related sequences is indicated in Fig. 1. The comparison of the critical residues between Pkapy and human calcium-activated nucleotidase (H-CAN) demonstrated that the targeted residues are conserved to some extent (Fig. 1). This alignment revealed that 4 of the 6 residues important for Ca 2+ binding (i.e., S168, E284, S345, E396), 4 of the 13 residues important for binding to nucleotides (i.e.,S168, E284, S345, E396), and 1 of the 3 residues essential for nucleotidase activity in H-CAN (i.e., D181) were conserved in Pkapy (Fig. 1). The 8 highly conserved regions among apyrases from vertebrates and invertebrates species, which had been introduced by Failer et al. were also conserved in Pkapy 25 . The amino acid homology and the similarity percentages between Ppapy and Pkapy proteins were 48.2% (163/338) and 68.0% (230/338), respectively.
The tertiary structure predictions and superimpositions. Prediction of the tertiary structure of Pkapy and Ppapy using SWISS-MODEL according to the homology modeling and QMEAN and GMQE scores revealed that both aforementioned apyrases have a close topology to homo-dimer of the human calcium-activated nucleotidase (H-CAN; accession N o .: 2H2N PDB). Thus, H-CAN was considered as the reference molecule in superimposition and to find the counterparts of the structurally-important residues. The structural superimpositions of Pkapy and Ppapy with H-CAN revealed that they are very similar in structure, especially in critical residues and functional domains (Fig. 2a-c). Notably, a comparison of RMSDs of Pkapy and Ppapy with the reference molecule indicated that the subtraction RMSDs values of the reference and the target molecules at specific positions are less than 0.5 Å which shows the backbone similarity of the reference and Pkapy and Ppapy molecules (Table 3). In addition, analysis of the torsional angles (Ramachandran plot) to validate the 3D modeled structures of Pkapy and Ppapy showed 89.5% and 89.3% of the residues are in the favored regions, respectively (Fig. 2d,e). The superimpositions and comparisons of the critical residues of the active site revealed that the targeted residues are completely similar to their counterparts.

Prediction and comparison of B cell epitopes of Pkapy and Ppapy. The linear B cell epitopes of
Pkapy and Ppapy were predicted using Bepipred 2.0 server (Table 4). Based on the scores of the default thresholds, 8 and 10 linear sequences were designated as B cell epitopes of Pkapy and Ppapy, respectively (Additional file 2: Fig. S2). The profiles of the antigenic peptides were very similar together and were located in the same topological position in the first structure of the two proteins.
The discontinuous B cell epitopes were estimated using ElliPro, based on the 3D structure of the proteins. The protrusion index (PI) of the residues was calculated to show the residues' solvent accessibility. Higher scores were determined as larger solvent accessibility of the residues. The PI values (above the default 0.5 scores) of discontinuous B cell epitopes for Pkapy and Ppapy are reported in Supplementary Table S1. Seven discontinuous B cell epitopes with similar topological positions for each protein were predicted.
Expression and purification of recombinant Ppapy and Pkapy. The recombinant Ppapy and Pkapy proteins were successfully expressed in E. coli and a high level of purification was achieved by Ni-NTA affinity chromatography. SDS-PAGE and Western blotting of the purified recombinant Ppapy and Pkapy indicated distinct bands with ~ 39 kDa as the expected Mw of the recombinant proteins ( Fig. 3a,b, Supplementary Fig. S3).

Antibody responses. The antibody responses to and cross-reactivity between the recombinant Ppapy and
Pkapy were investigated. Dot-blot results indicated that both proteins were immunogenic in BALB/c mice and induced antibodies against respective recombinant proteins. Besides, the antibodies generated in Ppapy-or Pkapy-immunized mice sera could react with both Ppapy and Pkapy but not with the control (BSA; Fig. 4a). To verify whether the recombinant proteins have conserved their immunogenic properties after expression in a prokaryote system, SGL from P. papatasi was spotted on a nitrocellulose membrane and the reactions with sera containing anti-Ppapy, and anti-Pkapy antibodies and normal mouse serum (as a negative control) were tested. Notably, antisera produced against the recombinant apyrases reacted with SGL from P. papatasi (Fig. 4b), indicating that antibodies against both recombinant proteins could react with native apyrase from P. papatasi. The ELISA results confirmed the immunogenicity, and cross-reactivity of the antibody responses in mice immunized with Ppapy and Pkapy. Sera from Ppapy-immunized mice reacted with Ppapy protein significantly stronger, compared to Pkapy and BSA (p < 0.0001). Likewise, sera from Pkapy-immunized mice reacted with Ppapy protein significantly stronger compared to Ppapy and BSA (p < 0.0001). The significantly higher reactivity of anti-Ppapy with Pkapy, and anti-Pkapy with Ppapy compared to reactivity with BSA (p < 0.05) was indicative of the specificity of the antigen-antibody reaction. The mean OD value for reactivity of anti-Ppapy sera with Ppapy protein was 1.4 while for the same amount of Pkapy-coated-wells, the mean OD value was 0.2. On the other hand, the OD values for a reaction between anti-Pkapy sera and Pkapy and Ppapy proteins were 1.6 and 0.3, respectively (Fig. 4c). These results demonstrated an average of 18.8% reactivity of anti-Pkapy sera with Ppapy protein while anti-Ppapy sera showed 14    T cell proliferation and cytokine assay. Two weeks after the last booster, Alamar Blue was used as a sensitive test to measure the proliferation of lymphocytes in a recall response, quantitatively. The results showed that both groups of immunized mice had recall responses to the relevant recombinant apyrases. Meanwhile, no stimulation occurred with the irrelevant protein. Likewise, the splenocytes of the unimmunized mice did not respond to the recombinant apyrases. The proliferative responses of the lymphocytes from both of the immunized groups to Ppapy or Pkapy were almost similar (Fig. 5a). The concentration of secreted IFN-γ, IL-4, and IL-10 upon in vitro stimulation of the spleen cells with Pkapy was determined by ELISA. The mice immunized    Fig. 5b), and IL-10 (p < 0.01, Fig. 5d), compared to the unimmunized mice. There was no statistically significant increase in IL-4 secretion by Pkapy-immunized mice (Fig. 5c).

Discussion
VL is endemic in different parts of Iran and P. kandelakii is one of the most reported vectors of the disease in the North-West and North-East provinces of the country 5,6,26 . P. kandelakii has been found to be naturally infected with L. infantum in Iran and Georgia 27 . So far, salivary cDNA libraries from 9 species of Phlebotomus genus have been constructed and different protein families have been identified 4 ; however, there is no study on the identification and characterization of salivary proteins from P. kandelakii. Apyrase is one of the prominent proteins of sand flies' saliva, with the potentials as a marker of exposure and as a vaccine component against leishmaniases.
To the best of our knowledge, there are two studies related to the cell-mediated responses against apyrases, as follows. It has been shown that DNA plasmid encoding P. ariasi salivary apyrase (ParSP01) induces a specific DTH response to the SGL from P. ariasi 21 . Furthermore, transfection of PBMC with a plasmid coding for Ppapy has resulted in a Th1 cellular immune response 22 . This designates the development of cell-mediated immune response by the apyrases and represents them as promising anti-Leishmania vaccine candidates. There are more studies on antibodies against apyrases; in fact, anti-apyrase antibodies have been considered as a potential biomarker of sand fly exposure and a risk factor for Leishmania transmission as has been shown in the case of canine exposure to P. perniciosus bites 15 .
In the present study, a full-length nucleotide sequence of P. kandelakii apyrase was characterized for the first time. Here, in contrast to many other studies that had identified the salivary proteins through the construction of cDNA libraries, we used a Genome-Walking nucleotide sequencing technique to complete the upstream and downstream sequences of a partially available middle part of P. kandelakii apyrase from Iran. The analysis of the coding sequence of Pkapy gene indicated that it belonged to the Cimex family of apyrases. Protein sequence alignments showed considerable homology with the other apyrases of the Cimex family and their vertebrate homologs, namely CAN. Bioinformatics analyses revealed that Pkapy like other known apyrases of Phlebotomus sand flies is a ~ 36 kDa protein. Besides, the protein is hydrophilic and stable with one N-glycosylation susceptible site (N247), which makes it an appropriate candidate for vaccine development.
Characterizing the diversity and degrees of homology between salivary proteins from various sand flies is an important issue. P. papatasi is a proven vector of L. major in Iran and the most abundant sand fly species in the endemic areas of the country 23 . In an attempt to identify sand fly proteins that can be used as a global or general anti-Leishmania vaccine, we compared the in-silico characteristics of Ppapy with those of Pkapy. Moreover, the potential antigenic cross-reactivities of these two proteins were explored. Ppapy was found to be a hydrophilic  www.nature.com/scientificreports/ and stable molecule with two N-glycosylation susceptible sites (i.e., N17 and N209), compared to Pkapy which had only one N-glycosylation susceptible site (N247). The RMSD values of the structurally-important residues of Pkapy and Ppapy and the Ramachandran plots indicated that both proteins were structurally similar to H-CAN as the reference molecule. The superimpositions and comparisons of the critical residues of the active sites revealed that the targeted residues are completely similar to their counterparts. The in silico analyses of the B cells epitopes showed that most of the predicted epitopes were conserved between Ppapy and Pkapy; although, Ppapy exhibits two more linear epitopes. The profiles of the antigenic peptides were very similar together since the epitopes were located in the same topological positions, based on the primary structures of both proteins. Seven discontinuous B cell epitopes with similar topological positions for each protein were also predicted.
The obtained results also indicated that the recombinant Pkapy and Ppapy proteins were immunogenic in BALB/c mice. Considering the similarities found by the bioinformatics analyses, we examined the antigenic cross-reactivity between Pkapy and Ppapy, by dot-blot and ELISA analyses. Cross-reactive antibodies and crossprotection have been reported between closely-related species of Phlebotomus subgenus i.e. P. papatasi and P. duboscqi 28 . However, P. papatasi and P. kandelakii are not closely-related species and this might be the reason for the low cross-reactivity between Pkapy and Ppapy. Our dot-blot data also showed that SGL from P. papatasi could be recognized by anti-recombinant Ppapy and Pkapy antibodies. These findings suggest the conservation of epitopes of the native apyrase in the recombinant proteins expressed in E. coli. This was in line with the findings that antibodies against P. perniciosus saliva reacted with the recombinant apyrases (i.e., rSP01 and rSP01B) 17,18,29 .
We also investigated the cellular immune response to Ppapy and Pkapy by assessment of their proliferative responses. These results indicated that splenocytes from Ppapy-and Pkapy-immunized mice could specifically recall responses to their homologous proteins. In addition, cross-reactivity was documented between Ppapy and Pkapy, since both groups of immunized mice responded to both proteins. Evaluation of the cytokines profile after stimulation with Pkapy showed higher levels of IFN-γ, in mice immunized with Pkapy, compared to the unimmunized mice. These findings indicated that the immunity to Pkapy is a Th1-dominant response. Th1 response to saliva components at the site of inoculation may activate the infected macrophages, leading to the killing of the parasites during the early phase of the infection, and may also promote a faster Leishmania-specific Th1 response 4 . Increased production of IL-10 by murine macrophages in response to SP01 (apyrase from P. perniciosus saliva) is also in line with our finding. Altogether, the immunogenic properties of Pkapy with respect to BALB/c mice highlight its potential as a component for an anti-Leishmania vaccine.

Conclusions
This is the first report on the characterization of an apyrase from P. kandelakii, an important vector of L. infantum in Iran. Immunologic studies indicated that Pkapy and Ppapy are immunogenic in BALB/c mice and show crossreactive responses. Moreover, cytokine analysis of the immunized mice revealed a Th1-type response to recombinant Pkapy. These results are providing insights into Pkapy as a potential candidate for a vector-based vaccine.

Methods
Sand flies. Phlebotomus kandelakii sand flies were captured in Bojnurd area of North Khorasan Province (North-East of Iran) using CDC traps and sticky papers. The sand flies were identified based on external and internal morphological characteristics of the head and abdominal terminalia (i.e., pharyngeal and spermathecal characteristics). The genomic DNA samples from 5 female sandflies were extracted and pooled. Female P. papatasi (originating from Turkey), was kindly provided by Professor Petr Volf (Department of Parasitology, Charles University, Czech Republic via infravec2; grant agreement No 731060). Salivary glands of P. papatasi were dissected out under a stereo microscope into PBS, and were disrupted by three freeze/thaw cycles. Salivary homogenates were centrifuged at 10,000 × g for 3 min and the supernatants were used for the experiments.

Determination of the 5′-and the 3′-end sequences of P. kandelakii apyrase gene by
Genome-Walking method. Since at the beginning of this study, only a partial middle part of Pkapy gene was publicly available, a Genome-Walking sequencing technique was performed to obtain the upstream and downstream sequences of the available segment, known as P. kandelakii clone kandG2a apyrase-like protein (GenBank accession N o .: JF899992). As shown in Table 1a, gene-specific primers (i.e., Forwards: F-GSPa, F-GSPb, and F-GSPc; Reverses: R-GSPa, R-GSPb, and R-GSPc) were designed and synthesized (Cinnaclon, IR-Iran) according to the 3′-end and the 5′-end sequences of JF899992 fragment using Gene Runner software (version 5.1.06 Beta). The specificity of the primers for PCR was checked by nucleotide BLAST on NCBI.
Seven Genome-Walking primers (i.e., GWPs A-G), as well as long and short universal tagging primers (i.e., UAP-N1, UAP-N2), were used according to the previous studies 24,30 . Firstly, to amplify the targeted sequences downstream of the gene, the synthesis of single-stranded DNA (ssDNA) molecules was evaluated using F-GSPa primer at 57-65 °C using a thermocycler (Mastercycler gradient 5331, Eppendorf, Germany). The products were electrophoresed on 1% agarose gel and analyzed. The lowest temperature in which no amplicon was seen was selected as the best temperature for further ssDNA molecule synthesis. The synthesis was performed separately in 7 tubes labeled A to G. Each tube was allocated for one of the GWPs in the following step. Amplification reactions were composed of 7.5 μl ExPrimeTaq Premix 2X (Genet Bio, South Korea), 400 nM F-GSPa primer, and sterilized D.W. up to 15 μl. The PCR program is mentioned in Table 1b.
Secondly, after immediately adding 1 μl from each of the 7 GWPs, along with 3 μl ExPrimeTaq Premix and 1 unit of Taq DNA polymerase (Genet Bio, South Korea) individually to the 7 reaction tubes, PCR was carried out according to the program indicated in Table 1b. Thirdly, 1 μl of 25-fold diluted PCR products was used as a template with UAP-N1 and F-GSPb primers for the first nested PCR as mentioned in Table 1b www.nature.com/scientificreports/ PCR products were diluted (25-fold) and 1 μl of each reaction was used as templates for the second nested PCR with F-GSPc and UAP-N2 primers, performed each time in 7 separate tubes using the same above-mentioned program (Table 1b). The amplicons of the final step were evaluated by 1.5% agarose gel electrophoresis. The fragments with acceptable sizes were subjected to gel extraction and were then subcloned into a PTZ57R/T (Thermo Scientific InsTAclone PCR Cloning Kit, USA) and sequenced (Macrogen, South Korea). To identify the upstream sequence of the gene, all the above-mentioned procedures were carried out, except that the specific reverse primers were used.
Tertiary structure prediction and superimposition. SWISS-MODEL server (http:// swiss model. expasy. org) was employed to predict the 3D structure of Pkapy and Ppapy based on homology modeling. According to QMEAN (Qualitative Model Energy Analysis) and GMQE (Global Model Quality Estimation), the best-predicted structures with the highest score for each protein were selected and used for comparing the structural properties. The superimposition and root-mean-square deviation (RMSD) analyses were performed by DeepView/Swiss-PdbViewer v.4.1.0 and UCSF Chimera v.1.11.2 33 .
Prediction of B cell epitopes. Linear B cell epitopes of Pkapy and Ppapy were predicted using BepiPred-2.0 of the IEDB analysis resource (http:// tools. iedb. org/ bcell/). The BepiPred-2.0 server predicts B-cell epitopes from the protein sequence, where the residues with scores above the default value of 0.5 were predicted as epitopes and colored in yellow on the graph. Further, the discontinuous B cell epitopes were predicted using Ellipro method in the IEDB database (http:// tools. iedb. org/ ellip ro/) which is based on solvent accessibility and flexibility 34 . Cloning, expression, and purification of recombinant Pkapy and Ppapy. Constructs containing regions encoding each protein without the putative secreted signal peptide were optimized for expression in E. coli and synthesized by Biomatik Corp. (Canada), subcloned into pET-21b (+) vector for expression with a C-terminal 6 × His-tag. The constructed expression vectors were transformed into E. coli BL21 (DE3; Invitrogen, USA) and verified by restriction digestion and nucleotide sequencing. Protein expression was induced by the addition of Isopropyl β-d-1-thiogalactopyranoside (IPTG) to a final concentration of 0.5 mM to a culture of LB with ampicillin (100 μg/ml), containing verified E. coli BL21 with the construct, grown to OD 600 ~ 0.5 (37 °C,200 RPM). The culture was grown for an additional 2 h after the induction as above.
The expressed recombinant proteins were purified by Ni-NTA Superflow resin (Qiagen, Germany), following the instructions under denaturing conditions. The proteins were dialyzed overnight against several changes of PBS and endotoxins were removed using a Pierce High-Capacity Endotoxin Removal Resin spin column (Thermo Scientific, USA), according to the manufacturer's recommendations. The concentrations of the purified recombinant proteins were determined by Bradford assay 35 .

SDS-PAGE and Western blotting analyses.
The purified recombinant Ppapy and Pkapy proteins were subjected to 10% SDS-PAGE, then electroblotted onto PVDF membrane using a wet Bio-Rad transfer system (Bio-Rad, Hercules, USA). The membrane was blocked with 5% (w/v) skimmed milk in PBS and incubated with anti-His antibody (Qiagen, Germany) as the primary antibody. Finally, the membrane was incubated with goat anti-mouse IgG HRP antibody (Sigma, Germany) as the secondary antibody. The reactivity was detected using 3, 3-diaminobenzidine tetrahydrochloride (DAB).
Immunization with the recombinant apyrases. Female BALB/c mice (4-6 weeks old) were purchased from the animal facility of the Production Complex of the Pasteur Institute of Iran. All animal studies were performed in line with the ARRIVE reporting guidelines. Approval was granted by the Ethics Committee of the Pasteur Institute of Iran (IR.PII.REC.1395.109), and the experiments were performed in accordance with relevant guidelines and regulations.
Mice (4 in each group) were immunized three times at 2-week intervals subcutaneously at the base of the tails with 10 µg of either Ppapy or Pkapy along with 15 µg Quil A (Invivogen, USA), as an adjuvant. The optimal dose of proteins for immunization was determined in a preliminary study in which mice were immunized with 5, 10, and 15 µg of Pkapy. When the sera were checked for the presence of anti-apyrase antibody, the results www.nature.com/scientificreports/ of 15 µg Pkapy were almost similar to 10 µg; hence, the latter amount was selected for the immunization. The control group received Quil A only. Mice were retro-orbitally bled for sera preparation two weeks after the last immunization.

Dot-blot immunoassay and ELISA.
To detect immunogenicity and the potential cross-reactivity of the recombinant apyrases, dot-blot and ELISA were performed. For the dot-blot assay, 1 μg of each Ppapy, Pkapy, SGL (1 gland/dot) of female P. papatasi, and an unrelated protein (i.e., BSA as a negative control) were spotted onto a nitrocellulose membrane. The spots were dried and the membrane was blocked with 5% skimmed milk in PBS. The membrane was then incubated with 1:10 diluted sera collected from the immunized and unimmunized mice for 2 h at RT. For the secondary antibody, an HRP-conjugated goat anti-mouse antibody was added and incubated at RT for 1 h. Finally, spots were developed using DAB substrate solution in presence of hydrogen peroxide. Stained dots on a white background indicated positive results. Ppapy, Pkapy, and BSA were used in 1 μg/well concentration to coat 96-well ELISA plates (Greiner, Germany) in duplicates and were then incubated at 4 °C overnight. After washing, pre-diluted sera (1:100) from each mouse (4 mice in each group) were added to the wells in a direct and cross manner to evaluate its reactivity with the immunized protein and the counterpart protein. After incubation for 2 h at RT, HRP-conjugated goat anti-mouse antibody (Sigma, Germany) was added to the plates and incubated for 1 h at RT. The plates were developed with 3,3′,5,5′-Tetramethylbenzidine (TMB; Sigma, Germany) and read at 450 nm by a microplate reader (Biochrom Anthos 2020, UK). The levels of IgG antibody were reported as OD values of each well minus the OD values of the control wells.
T-cell proliferation and cytokine assay. Alamar blue assay was used to evaluate the proliferative responses of the lymphocytes to the recombinant proteins. Two weeks after the last immunization, the spleen cells from each mouse (4 mice in each group) were recovered and cultured as previously described 36 . In brief, the splenocytes were stimulated with recombinant Pkapy, Ppapy, or an irrelevant recombinant protein (FHA from Bordetella pertussis). Moreover, the splenocytes of unimmunized mice were stimulated with the recombinant apyrases. Negative control cultures consisted of the medium without any antigen. The cultures were incubated at 37 °C, 5% CO 2 for 3 days. Subsequently, 20 μl of Alamar blue reagent (Sigma, Germany; 0.15 mg/ml in PBS) was added to each well and incubated for 4 h. Three replicates of each sample were analyzed on a microplate reader at 570 nm with a reference wavelength of 690 nm (BioTek ELx808 Absorbance Microplate Reader, USA) and the percentage of Alamar Blue reduction was calculated for stimulated and unstimulated cells according to Al-Nasiry et al. 37 . The stimulation index (SI) was calculated by dividing the mean results of the stimulated cells by the mean results of the un-stimulated cells. For cytokine assays, splenocytes from Pkapy-immunized mice were stimulated with recombinant Pkapy for 4 days and subsequently, the culture supernatants were recovered and stored at − 80 °C for determination of IFN-γ, IL-4, and IL-10 levels. Cytokines were assessed by Mabteck ELISA kits (Stockholm, Sweden), according to the manufacturer's instructions. Statistical analysis. ANOVA followed by Tukey's multiple comparisons test and Student's t-test were used to evaluate the statistical significance of the obtained data for ELISA and T cell proliferation, respectively. Statistical analysis was performed with GraphPad Prism software (Prism 8.0.2., 2019, San Diego, CA). The p < 0.05 was considered to be significant, and data were represented as mean + SD.

Data availability
The datasets generated and analyzed during the current study are available in the repository, NCBI GenBank repository, Accession N o .: MN893300.